Increasingly, autophagy is recognized as being involved in apoptosis. Autophagy is the mechanism by which cells rid themselves of damaged, worn out, and surplus organelles. In liver, glucagon and nutrient withdrawal are physiologic inducers of autophagy. Our preliminary experiments indicate that i) glucagon and nutrient withdrawal stimulates mitochondrial depolarization, ii) depolarized mitochondria move into acidic lysosomal/autophagosomal compartments, and iii) cyclosporine A (CsA) blocks both mitochondrial depolarization and lysosomal proliferation. Accordingly, a central hypothesis of this project is that the MPT is a crucial step signaling and initiating mitochondrial autophagy. Our overall goal is to understand the signaling pathways and mechanisms of MPT-mediated autophagy in cultured rat hepatocytes. Specific Aim 1 is to test the hypothesis that the mitochondrial permeability transition (MPT) precedes and signals mitochondrial autophagy using fluorescent indicators to monitor directly mitochondrial polarization, mitochondrial membrane permeability, and movement to mitochondria into acidic autophagosomes by confocal and multiphoton microscopy. Specific Aim 2 is to characterize the factors regulating mitochondrial autophagy. In isolated formation of reactive oxygen species (ROS) promote onset of the MPT. Ceramides, gangliosides and pro-apoptotic Bcl-2 family members also promote the MPT. Therefore, we will determine by confocal and multiphoton microscopy what alterations of mitochondrial NAD(P)H, GSH, free Ca2+, and ROS formation precede mitochondrial autophagy after stimulation with glucagon and nutrient deprivation. Specific Aim 3 will examine the mechanistic relationships between autophagy and apoptosis. Since the MPT appears to mediate both autophagy and apoptosis, we will determine whether specific apoptotic signals (e.g., TNFalpha, Fas ligation) stimulate autophagy in hepatocytes, and whether stimulation of autophagy augments apoptosis. Similarly, we will assess whether inhibition of autophagy inhibits apoptosis and whether pro-apoptotic caspases in the initiation or progression of autophagy. Over all, these experiments will test the hypothesis that apoptosis begins to occur when onset of the MPT in increasing numbers of mitochondria exceeds the capacity of autophagy to sequester pro-apoptotic signals released after the MPT and confine lysosomal enzymes within autophagosomes.